Download Summary Colorectal cancer (CRC) is a major health problem

Summary
Colorectal cancer (CRC) is a major health problem. With regard to the prevalence
of cancer disease in Poland, CRC is currently in the third place in both men and women.
In addition, a steady trend of increasing incidence of this cancer has been noticed.
So far conducted microscopic examinations concerning alterations of enteric nervous system
(ENS) plexuses’, in the vicinity of the infiltration of the colon wall by the CRC tumor,
revealed morphological changes of plexuses as well as the changes in the expression
of various neuropeptides including galanin. Human galanin (Gal) is a 30-amino acid
neuropeptide that is present in the central and peripheral nervous systems and in peripheral
tissues. The presence of Gal and its receptors has also been demonstrated in the CRC tumor
tissue, suggesting a role of Gal as a biomarker of CRC development and progression.
Moreover, a significant increase of Gal serum concentration was observed in a group
of Korean CRC patients as compared to healthy individuals.
The primary objective of the research was to determine Gal serum concentration
in the Polish population of CRC patients and to measure the content of Gal in particular layers
of the large intestine wall (containing ENS plexuses) and, separately, in the CRC tumor tissue.
The immunohistochemical technique was applied to examine the presence of Gal in particular
layers of large intestine wall containing ENS plexuses and in the tumor tissue and to perform
the morphometric analysis of the observed morphological changes of the ENS plexuses
in the sections of colon wall located close to and distant from the CRC tumor. Furthermore,
the mechanisms leading to the observed morphological changes of peritumoral ENS plexuses
were investigated.
Full blood samples were collected preoperatively from 68 CRC patients
who underwent surgery at the Warmia and Mazury Oncological Center in Olsztyn (Poland)
and a control group of 39 healthy volunteers. The postoperative colorectal samples were
harvested from 31 patients. Tissue homogenates were prepared from separated parts
of the intestinal wall. By means of enzyme-linked immunosorbent assay (ELISA)
the concentration of Gal in the sera of CRC patients and healthy volunteers as well as Gal
content in the homogenates of the particular layers of the colon wall and CRC tissue were
determined. From the full-thickness large intestine wall samples, 5 µm thick paraffin sections
were prepared, in order to detect Gal immunoreactivity and the presence of inflammatory
cells (neutrophils and macrophages) and on the cryostat sections triple immunofluorescence
staining was performed in order to visualize the coexpression of caspases 3 and 8 (CASP 3
and CASP 8) and Gal.
The mean Gal concentration in the sera of CRC patients was 119.47 pg/ml
and was significantly higher than the mean Gal serum concentration in healthy volunteers 50.14 pg/ml. The highest levels of Gal were identified in the homogenates obtained from
tumor tissue and mucosa from the non-cancerous section of the intestinal wall, 408.71 ± 30.28
pg/g and 462.73 ± 28.54 pg/g, respectively. Gal content in the mucosa in the vicinity
of cancer infiltration was 306.63 ± 27.60 pg/g and was significantly lower than in the first two
locations. Gal content in the muscularis externa from the border of cancer infiltration was
286.66 ± 13.43 pg/g and was significantly higher than the Gal content in muscularis externa
from the control part of the large intestine. Morphometric analysis of the area occupied
by Gal-immunoreactive perikaryons in myenteric plexuses of 22 CRC patients revealed
that the mean area of Gal-immunoreactive myenteric plexuses in the intestinal wall proximal
to the CRC tumor was approximately 50% smaller than in the distant section of the colon
wall. Statistical analysis revealed no associations between Gal serum concentration, myenteric
plexuses’ atrophy as well as Gal content in particular layers of colon wall and cancer tissue
and clinicopathological features, including gender, age, tumor location, depth of invasion,
lymph node metastasis and TNM stage. Moreover, morphological analysis was carried
out in order to establish the proportion of CASP-3- and CASP-8-immunoreactive cells
in colocalization with Gal in the population of PGP 9.5-positive neurons. No differences
in the presence of CASP-8- and CASP-3-positive neurons between intestinal wall sections
located adjacent to or distant from the CRC tumor were found. The number of CASP-3
and Gal coexpressing neurons was 3-fold higher than the number of CASP-8 and Gal
coexpressing neurons. Since Gal exerts neuroprotective effects, it may be hypothesised
that within the myenteric plexuses, during CRC development and progression, an extrinsic
apoptotic pathway is induced with silencing of this process at the effector stage caused
by the presence of Gal (observed coexpression). In addition, the analysis of the presence
of inflammatory cells - macrophages and neutrophils, did not show their accumulation
within or near the myenteric plexuses located in the proximity of CRC tumor, excluding their
possible contribution to the plexuses’ atrophy.
In conclusion, a higher Gal serum concentration in the population of Polish CRC
patients when compared to healthy subjects was found. A significantly higher Gal content
in the cancer tissue than in the myenteric plexuses together with high Gal immunoreactivity
within cancer cells suggests that the tumor tissue may be a significant source
of this neuropeptide in blood of CRC patients despite evident atrophy of ENS plexuses
localized in the vicinity of the CRC tumor. These observations suggest a potential role
of Gal as a serum and tissue CRC biomarker. The results of IHC and immunofluorescence
stainings, carried out in the second part of the study, excluded inflammation and necrosis
as well as apoptosis as the causes of myenteric plexuses’ atrophy in the vicinity of CRC
tumor.